SUMMARY The Na+-pumping NADH dehydrogenase (Na+-NQR) is the main ion pump, and the entry point of electrons into the respiratory chain of diverse types of pathogens, such as Vibrio cholerae, Pseudomonas aeruginosa, Chlamydia trachomatis, etc. This complex is essential in bacterial physiology, producing a sodium gradient that sustains many critical processes, including nutrient transport, pH regulation, ATP synthesis, virulence factor secretion, as well as drug elimination. Remarkably, the Na+-NQR family has evolved separately from the main families of ion transporters and redox enzymes, and employs novel enzymatic mechanisms not found elsewhere. Recently, our group proposed a comprehensive model for its catalytic mechanism that incorporates all available functional and structural data. Our model indicates that ion pumping follows a completely novel mechanism, in which the energy released by the redox reactions produces conformational changes that drive ion uptake and transport. The main aim of this project is to understand the role of one-electron reactions and flavin radicals in the mechanism of sodium transport by Na+-NQR, and the location and role of riboflavin and a newly identified Fe center in electron transfer. Na+-NQR is the only reported enzyme that is able to use riboflavin as a redox cofactor. Moreover, this molecule is found as a neutral radical with a critical role in sodium transport. Our preliminary data indicates that the riboflavin binding site is a completely a new structural motif, located in the interface of subunits B, E and D. In this project we will map the Riboflavin binding site, and will study the mechanisms that allow electron transfer and the stabilization of the neutral flavin radical. In addition, we will characterize Thioridazine as a new inhibitor that targets this site, which could allow the development of a new class of antibiotics against this enzyme. Moreover, we will study the role of a recently reported Fe center in the electron transfer chain, as the cofactor that could link the redox reactions in the cytosol with the reactions in membrane and periplasmic sides. To understand the role of one-electron transitions in the catalytic mechanism of the enzyme, the structural factors that stabilize the unpaired electrons in radical flavins will be identified. Mutants of conserved residues in the vicinity of the cofactors will be characterized, to understand the effect of the mutation on the properties of the cofactor, such as its midpoint potential and redox transitions. Moreover, the effect of these mutations will be evaluated over the activity of the enzyme, electron transfer chain and on ion transport process, to characterize the role of the radicals in different steps of the catalytic cycle. These studies are essential to understand the strategy used by the Na+-NQR family to transform the energy released by the redox transitions into molecular movements that drive ion transport, and the general underlying principles governing enzyme function and biological membrane transport. Moreover, these results are important in the current efforts develop antibiotics against this promising drug target.